Journal of Applied Genetics 46(1), 2005, pp. 75-83
Isolation, cloning and expression of DNA fragments reflecting open reading frames of 1.8 and 4.6 kb RNAs of the I-18 C gene of Chironomus tentans
Boguslaw P. Borowicz
Abstract: This study, based on the genetic engineering approach, concerns the regulation of the insect gene expression, but it has also a strong significance for plant protection, which is associated with the future possibility to control insect development more effectively, especially in those species that are pests of cultivated plants. The I-18 C gene of Chironomus tentans is activated by heat-shock and the steroid hormone - ecdysone. This gene produces different transcripts (at least four) by alternative splicing. Two different open reading frames, i.e. ORFs I and II of the two main transcripts of this gene, i.e. the 1.8 and 4.6 kb RNA, had been isolated, at the DNA level, as the ORFs reflecting DNA fragments, using the polymerase chain reaction, then were cloned into the bluescript vector and finally were cloned into the pET-3a vector to be translated in the T7 RNA polymerase/promoter system of E. coli. As the preliminary outcome of these experiments it was possible to obtain ORF I overexpressed as the polypeptide, whereas ORF II was expressed as the polypeptide that was strongly visible on SDS-polyacrylamide gel. This could facilitate further studies regarding the mechanisms of expression of this gene, including the level of expression of this gene during the C. tentans development.
Key words: alternative splicing, Chironomus tentans, cultivated plants protection, DNA cloning, open reading frames, potential insect control, T7 RNA polymerase/promoter system.
Correspondence: B.P. Borowicz, Institute of Natural Fibres, Wojska Polskiego 71b, 60-630 Poznan, Poland; e-mail: B.Borowicz@inf.poznan.pl
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